U.S. patent number 9,284,526 [Application Number 10/979,758] was granted by the patent office on 2016-03-15 for culture medium with yeast or soy bean extract as amino acid source and no protein complexes of animal origin.
This patent grant is currently assigned to GSK Vaccines S.R.L.. The grantee listed for this patent is Antonella Giglioli, Maria Kontakou, Roberto Olivieri, Rino Rappuoli, Fabio Sabbatini, Lucia Tagliaferri. Invention is credited to Antonella Giglioli, Maria Kontakou, Roberto Olivieri, Rino Rappuoli, Fabio Sabbatini, Lucia Tagliaferri.
United States Patent |
9,284,526 |
Olivieri , et al. |
March 15, 2016 |
Culture medium with yeast or soy bean extract as amino acid source
and no protein complexes of animal origin
Abstract
The present invention relates to a medium containing non-animal
derived proteinaceous material for cultivating patheogenic bacteria
to produce an immunogenic factor and processes employing such
medium.
Inventors: |
Olivieri; Roberto (Siena,
IT), Sabbatini; Fabio (Monteroni d'Arbia,
IT), Kontakou; Maria (Caserta, IT),
Tagliaferri; Lucia (Siena, IT), Giglioli;
Antonella (Siena, IT), Rappuoli; Rino
(Monteriggioni, IT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Olivieri; Roberto
Sabbatini; Fabio
Kontakou; Maria
Tagliaferri; Lucia
Giglioli; Antonella
Rappuoli; Rino |
Siena
Monteroni d'Arbia
Caserta
Siena
Siena
Monteriggioni |
N/A
N/A
N/A
N/A
N/A
N/A |
IT
IT
IT
IT
IT
IT |
|
|
Assignee: |
GSK Vaccines S.R.L. (Siena,
IT)
|
Family
ID: |
32180225 |
Appl.
No.: |
10/979,758 |
Filed: |
November 3, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050089968 A1 |
Apr 28, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10383001 |
Mar 7, 2003 |
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09424800 |
Feb 24, 2000 |
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PCT/IB98/00938 |
May 28, 1998 |
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Foreign Application Priority Data
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May 28, 1997 [GB] |
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9710981.3 |
Mar 30, 1998 [GB] |
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9806802.6 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
1/205 (20210501); A61K 39/02 (20130101); C12N
1/20 (20130101); A61P 31/04 (20180101); C12R
2001/21 (20210501) |
Current International
Class: |
C12N
1/20 (20060101) |
Field of
Search: |
;435/253.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2099632 |
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Jun 1993 |
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CA |
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0429816 |
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Jun 1991 |
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EP |
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0540897 |
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Sep 1992 |
|
EP |
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5759510 |
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Dec 1993 |
|
EP |
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0624376 |
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Nov 1994 |
|
EP |
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9409115 |
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Apr 1994 |
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WO |
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WO 94/09115 |
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Apr 1994 |
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WO |
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96/13576 |
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May 1996 |
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WO |
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96/14086 |
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May 1996 |
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WO |
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9614393 |
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May 1996 |
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WO |
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glutamine and asparagine by Helicobacter pylori," Journal of
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applicant .
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polysaccharide production by H. influenzae type b," J Chem Technol
Biotechnol 81:182-188. cited by applicant .
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Haemophilus parainfluenzae," Microbiology 27(1): 51-60. cited by
applicant .
Corbel (1994). "Control testing of combined vaccines: a
consideration of potential problems and approaches," Biologicals.
22(4):353-60. cited by applicant .
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Haemophilus influenzae from clinical and normal flora sources," J
Gen Microbiol. 113(2):409-11. cited by applicant .
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as diphtheria? Lessons to be relearned," Lancet. 348(9037):1289-92.
cited by applicant .
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toxin in submerged culture in relatively simple equipment using a
semisynthetic medium," Biotechnol Bioeng Symp. 0(4-1):283-93. cited
by applicant .
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growth and toxin production of Corynebacterium diphtheriae," J
Egypt Public Health Assoc.60(1-2):113-26. cited by applicant .
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Bacteriol. 85(1): 253-254. cited by applicant.
|
Primary Examiner: Afremova; Vera
Attorney, Agent or Firm: Lee; Helen Campen; Virginia
Claims
The invention claimed is:
1. A medium for cultivating Haemophilus influenzae to produce an
immunogenic factor wherein the medium comprises a Haemophilus
influenzae bacteria and at least about 50% by dry weight of a
non-animal derived proteinaceous material, and which does not
comprise animal derived proteinaceous material, wherein the
non-animal derived proteinaceous material is a soybean derived
protein composition.
2. A process for preparing an immunogenic factor of the Haemophilus
influenzae bacteria comprising the steps of cultivating the
Haemophilus influenzae bacteria in a medium comprising at least
about 50% by dry weight of a non-animal derived proteinaceous
material, wherein the medium does not comprise animal derived
proteinaceous material and the non-animal derived proteinaceous
material is a soybean derived protein composition.
3. The method of claim 2, further comprising purifying the
immunogenic factor from the medium.
4. A process for the production of a vaccine comprising preparing
an immunogenic factor of Haemophilus influenzae by the process of
claim 3, and bringing said factor, optionally toxoided, into
association with a pharmaceutically acceptable carrier.
Description
The present invention relates to a medium for cultivating
pathogenic bacteria. The present invention also relates to the use
of the medium to cultivate pathogenic bacteria, obtaining
immunogenic factors from the bacteria being cultivated and
preparing vaccines using the immunogenic factors.
Bacterial vaccines are produced by cultivating pathogenic bacteria
in a medium, isolating immunogenic factors and preparing vaccines
based on the isolated immunogenic factors. Such methods are
described in Bacterial Vaccines, 1984, Ed. Rene Germanier Academic
Press and Bacterial Vaccines in Advances in Biotechnology
Processes, Vol. 13, 1990, Ed. A. Mizrahi, Wiley-Liss. In
conventional methods, the pathogenic bacteria are cultivated in
media containing proteinaceous material of animal origin. These
compounds are used in the belief that some growth factors,
essential for the growth of pathogenic bacteria, were only present
in compounds of animal origin such as blood, brain heart infusion,
meat, etc. For instance, C. tetani is grown in media containing a
heart infusion and an enzymatic digest of casein; C. diphtheriae
requires a beef infusion; H. pylori is grown in media containing
pentamine and tryptone; and Haemophilus influenzae is grown in
media containing proteose peptones. World Health Organisation
report series numbers 800 (1990) and 814 (1991) indicate that to
grow Haemophilus influenza, Corynebacterium diphtheriae,
clostridium tetani and Bordetella pertussis, media comprising
compounds of animal origin are required.
The requirement for proteinaceous material of animal origin in the
media gives rise to concern over possible contamination of the
media. In particular, concern that the media may be contaminated
with the bovine spongiform encephalopathy (BSE) causative agent or
other infectious and harmful agents, restricts the usefulness of
any factors derived from such cultures, especially in therapeutic
applications.
It has surprisingly been found that proteinaceous materials of
non-animal origin are able to sustain the growth of pathogenic
bacteria and enable the production of immunogenic factors by the
bacteria.
In patent application DD 294 502-A, a process for preparing soya
hydrolysates and the use of the soya hydrolysates in culturing
microorganisms for fimbriae is disclosed. The soya hydrolysate is
prepared by culturing soya-flour-containing medium with
streptomyces strains. The soya hydrolysate is used as a carbon
source due to the high percentage of polysaccharides present and
contains less than 20% by dry weight protein.
The use of proteinaceous material of non-animal origin, such as
proteins from soy beans, cotton seeds, potatoes, etc., as a media
constituent for the cultivation of pathogenic bacteria, completely
removes the risk of animal derived contamination, such as BSE,
being transmitted into humans in any subsequent therapeutic or
prophylactic applications.
A further advantage associated with the use of vegetable derived
proteinaceous materials is the reduction in cost of producing the
materials and the increased consistency in the materials
(non-animal derived proteinaceous material is more uniform in its
composition than animal derived materials).
The present invention provides a medium for cultivating pathogenic
bacteria to produce an immunogenic factor wherein the medium
comprises at least 20% by dry weight of a non-animal derived
proteinaceous material, and does not comprise animal derived
proteinaceous material.
Any standard medium for cultivating bacteria may be used as the
basis for the medium of the present invention, provided the medium
does not contain animal derived proteinaceous material.
Preferably, the medium of the present invention comprises a carbon
and an energy source, a nitrogen source, essential salts and
optionally a selecting agent, such as an antibiotic, for selecting
the microorganisms to be cultured.
The medium of the present invention may be a solid or liquid
medium. Examples of standard liquid media which may form the basis
of the medium of the present invention include Brucella Broth
(without tryptone and peptamine), Watson medium (without casamino
acids), Mueller Miller medium (without heart infusion and casein
hydrolysate), CL medium (without casamino acids) and Franz A
medium. Standard solid media can be prepared from any of the liquid
media by the addition of a solidifying agent such as agar.
The term "cultivating" as used herein means the maintenance of, and
preferably the growth of, bacteria. Bacterial growth is herein
defined as an increase in bacterial biomass.
The term "pathogenic bacteria" as used herein, means any bacteria
which is involved in the pathogenesis of a disease. Preferred
pathogenic bacteria include Helicobacter pylori, Haemophilus
influenzae, Corynebacterium diphtheriae and Neisseria meningitidis,
Bordetella pertussis and Clostridium tetani.
The term "immunogenic factor" as used herein, means any factor
which is capable of stimulating the immune system of a human or
animal. Such immunogenic factors include antigenic proteins and
especially virulence factors and fragments thereof. Virulence
factors are defined as being associated with the virulence of a
bacteria and includes such factors as the vaculating cytotoxin VacA
produced by Helicobacter pylori. Other virulent factors are
described by Rappuoli, R. et al., European Journal of
Gastroenterology and Hepatology of Helicobacterpylori infection.
Proceedings of an interdisciplinary meeting (Genova, Jun. 18-19,
1993) J. J. Misiewicz, Ed. (CS Current Science), pp S76-S78
(incorporated herein by reference). The immunogenic factor may be
genetically detoxified or treated by a toxoiding process. Methods
for genetically detoxifying and toxoiding immunogenic factors are
well known to those skilled in the art and include those described
by Rappuoli, R., Vaccine, 12, 579-581, (1994) (herein incorporated
by reference).
The term "proteinaceous material", as used herein, means proteins
and protein degradation products including free amino acids.
Preferably, the proteinaceous material is a protein
hydrolysate.
Non-animal derived proteinaceous materials as used herein means
proteinaceous materials derived from non-mammalian sources, such as
vegetables, birds, fish, yeasts, funghi, algae and microorganisms.
More preferably, non-animal proteinaceous materials mean
proteinaceous material derived from vegetables, yeasts, algae and
microorganisms. Most preferably, non-animal derived proteinaceous
materials means proteinaceous materials derived from vegetables
such as protein compositions derived from soy beans, cotton seeds,
potatoes, etc.
Preferred non-animal derived proteinaceous materials include yeast
extracts such as HY YEST (Quest) and soy bean derived protein
compositions such as Hysoy (Quest), Amisoy (Quest), N-Z soy (Quest)
and Soytone (Difco).
Yeast extracts can be prepared by standard procedures well known to
those skilled in the art. Furthermore, yeast extracts are
commercially available from numerous sources including Sigma and
Quest.
Soy bean derived protein compositions can be prepared by enzymatic
digestion of soy bean meal or soy isolate using standard enzymes
such as papain. For example, N-Z soy is a soluble protein
composition made by the enzymatic digestion of a soy isolate and
Hysoy is a papaic digest of soy bean meal. Soy bean derived protein
compositions can also be obtained by acid hydrolysis of a soy
isolate. For example, Amisoy is a source of amino acids and
peptides produced by acid hydrolysis of a soy isolate.
Other non-animal derived proteinaceous materials can be obtained by
either enzymatic digestion or by acid hydrolysis of a protein
containing material of a non-mammalian source.
The non-animal derived proteinaceous material in the medium of the
present invention may comprise two or more different non-animal
derived proteinaceous materials, such as a mixture of soy bean
derived protein compositions such as Hysoy, Amisoy, N-Z soy and
Soytone.
Animal derived proteinaceous materials include protein compositions
such as fetal calf serum (FCS), bovine serum albumin (BSA),
proteose peptones, casamino acids, tryptone, peptamin and casein
hydrolyzates.
Preferably, the medium of the present invention comprises at least
20% by dry weight of a non-animal derived proteinaceous material,
more preferably, at least 30% by dry weight of a non-animal derived
proteinaceous material and most preferably at least 50% by dry
weight of a non-animal derived proteinaceous material.
It has surprisingly been found that the cultivation of pathogenic
bacteria using the medium of the present invention results in
increased growth of the bacteria and an increased yield of
immunogenic factors compared to cultivation of the pathogenic
bacteria in a medium containing animal derived proteinaceous
material.
The present invention further provides a process for making a
medium for cultivating pathogenic bacteria to produce an
immunogenic factor comprising adding sufficient non-animal derived
proteinaceous material to a standard medium for cultivating
bacteria, which does not comprise animal derived proteinaceous
material, so that the medium for cultivating pathogenic bacteria
comprises at least 20% by dry weight of the non-animal derived
proteinaceous material, and does not comprise animal derived
proteinaceous material.
Examples of standard liquid media include Brucella Broth (without
tryptone and peptamine) Watson medium (without casamino acids),
Mueller Miller medium (without heart infusion and casein
hydrolysate), CL medium (without casamino acids) and Franz A
medium. Standard solid media can be prepared from any of the liquid
media by the addition of a solidifying agent such as agar.
The present invention further provides a culture comprising the
medium of any of the previous claims and pathogenic bacteria.
Preferred pathogenic bacteria include Helicobacter pylori,
Haemophilus influenzae, Corynebacterium diphtheriae and Neisseria
meningitidis, Bordetella pertussis and Clostridium tetani. Most
preferably, the pathogenic bacterium is Helicobacter pylori.
The present invention also provides a process for preparing an
immunogenic factor of a pathogenic bacteria comprising the steps of
cultivating the bacteria in the medium of the present invention and
optionally purifying the immunogenic factor from the medium.
Preferably, the pathogenic bacteria are cultured in the medium of
the present invention for at least 6 hours, more preferably at
least 36 hours, and most preferably at least 72 hours under
suitable conditions for the production of the immunogenic
factor.
Suitable culture conditions for the production of the immunogenic
factor, including the duration of the culture, will vary depending
on the bacteria being cultured. However, one skilled in the art can
easily determine the culture conditions required for the production
of the immunogenic factor by following standard protocols, such as
those described in the series Methods in Microbiology, Academic
Press Inc., (incorporated herein by reference) and, if necessary,
by performing a number of standard experiments to determine
suitable culture conditions.
The immunogenic factor can be isolated from the bacterial culture
using a number of standard techniques including those described by
Manetti, R. et al., Infect. Immun., 63, 4476-4480, (1995),
incorporated herein by reference.
The present invention also provides a process for the production of
a vaccine comprising preparing an immunogenic factor of a
pathogenic bacteria comprising the steps of cultivating the
bacteria in the medium of the present invention, optionally
purifying the immunogenic factor from the medium and bringing said
factor, optionally toxoided, into association with a
pharmaceutically acceptable carrier. Suitable methods for producing
a vaccine are described by Rappuoli, R., New and improved vaccines
against Diphtheria and Tetanus. (1990), 251-268, New Generation of
Vaccines, Ed. G. C. Woodrow, M. M Levine, Marcel Dekker Inc. New
York. (Incorporated herein by reference).
The vaccines prepared by the process of the present invention will
require the addition of adjuvants when they are used. Suitable
adjuvants are described in Gupta, R. K. et al., Vaccine, 13,
1263-1276, (1995).
The vaccines prepared by the process of the present invention can
be used to vaccinate an individual against a bacterial infection.
Preferred bacterial infections which can be vaccinated against
include type B gastritis, bacterial meningitidis, diphtheria,
tetanus and whooping cough.
The vaccines prepared by the process of the present invention may
be provided as a pharmaceutical composition comprising the vaccine
of the present invention in admixture with a pharmaceutically
acceptable carrier and adjuvants as mentioned above.
The vaccines prepared by the process of the present invention can
be administered by oral or parenteral route, including intravenous,
intramuscular, intraperitoneal, subcuaneous, transdermal, airway
(aerosol), rectal and topical administration.
For oral administration, the compounds of the invention will
generally be provided in the form of tablets or capsules or as an
aqueous solution or suspension.
Tablets for oral use may include the active ingredients (the
vaccine component) mixed with pharmaceutically acceptable
excipients such as inert diluents, disintegrating agents, binding
agents, lubricating agents, sweetening agents, flavouring agents,
colouring agents and preservatives. Suitable inert diluents include
sodium and calcium carbonate, sodium and calcium phosphate, and
lactose, while corn starch and alginic acid are suitable
disintegrating agents. Binding agents may include starch and other
well known agents, while the lubricating agent, if present, will
generally be magnesium stearate, stearic acid or talc. If desired,
the tablets may be coated with a material such as glyceryl
monostearate or glyceryl distearate, to delay absorption in the
gastrointestinal tract.
Capsules for oral use include hard capsules on which the active
ingredient is mixed with a solid diluent, and soft capsules wherein
the active ingredient is mixed with water or an oil such as peanut
oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous
use, the compounds of the invention will generally be provided in
sterile aqueous solutions or suspensions, buffered to an
appropriate pH and isotonicity. Suitable aqueous vehicles include
Ringer's solution and isotonic sodium chloride. Aqueous suspensions
according to the invention may include suspending agents such as
cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and
gum tragacanth, and a wetting agent such as lecithin. Suitable
preservatives for aqueous suspensions include ethyl and n-propyl
p-hydroxybenzoate.
The vaccines prepared by the process of the present invention may
also be presented as liposome formulations.
The present invention is now described with reference to the
following examples and to the figures in which:
FIG. 1 shows the kinetics of growth of H. pylori CCUG 17874 in BB
containing Tryptone and peptamin.
FIG. 2 shows the kinetics of growth of H. pylori CCUG 17874 in
simplified BB containing Soytone.
FIG. 3 shows the kinetics of growth H. pylori CCUG 17874 in
simplified BB containing Hysoy.
FIG. 4 shows a VacA immunoblot wherein lane 1 is molecular weight
marker, lane 2 VacA standard 750 ng, lane 3 VacA standard 500 ng,
lane 4 VacA standard 400 ng, lane 5 VacA standard 150 ng, lane 6
VacA standard 20 ng, lane 7 VacA produced at the end of
fermentation, lane 8 VacA produced after 48 hours of culture, lane
9 VacA produced after 30 hours of culture, lane 10 VacA produced
after 23 hours of culture.
FIG. 5 shows a VacA immunoblot wherein lane 1 is molecular weight
marker, lane 2 VacA standard 750 ng, lane 3 VacA standard 500 ng,
lane 4 VacA standard 400 ng, lane 5 VacA standard 150 ng, lane 6
VacA standard 20 ng, lane 7 VacA produced at the end of
fermentation, lane 8 VacA produced after 31.5 hours of culture,
lane 9 VacA produced after 30 hours of culture, lane 10 VacA
produced after 24 hours of culture.
FIG. 6 shows a VacA immunoblot wherein lane 1 is molecular weight
marker, lane 2 VacA standard 750 ng, lane 3 VacA standard 500 ng,
lane 4 VacA standard 400 ng, lane 5 VacA standard 20 ng, lane 6
VacA produced at the end of fermentation, lane 7 VacA produced
after 48 hours of culture, lane 8 VacA produced after 31 hours of
culture, lane 9 VacA produced after 24.5 hours of culture, lane 10
VacA standard 150 ng.
FIG. 7 shows the kinetics of growth of H. influenzae b in
simplified Franz medium containing Soytone or Proteose peptone.
FIG. 8 shows the kinetics of growth of N. meningitidis C in Watson
medium containing Casaminoacids or Amisoy.
EXAMPLES
All references are herein incorporated by reference.
Example I
Helicobacter pylori is a curved gram-negative microaerobic
bacterium isolated about 10 years ago and is associated with type B
gastritis in humans. This bacterium colonizes the human gastric
mucosa and establishes a chronic infection that may result in
gastric and duodenal ulcers (Blaser, M. J., (1990), J. Infect.
Dis., 161, 629-633) and can be a risk factor for the development of
gastric carcinoma (Parsonnet, J. et al., (1991), New Engl. J. Med.,
325, 1127-1131).
In the long term, the infection and the diseases could be prevented
and treated by vaccination. Currently, several factors involved in
bacterial adhesion, colonisation and virulence have been
identified. One of the most interesting factors involved in the
disease is the vacuolating cytotoxin (VacA) that causes massive
vacuolization in several mammalian cell lines (Leunk, R. D.,
(1991), Rev. Infect. Dis., 13(suppl.8), S683-S689). Vacuoles have
also been observed in the gastric epithelia of patients with
chronic gastritis (Tricottet, V. et al., (1986), Ultrastruct.
Pathol., 10, 113-117). This protein has been shown to cause
ulceration in mice (Telford, J. L. et al., (1994), J. Exp. Med.,
179, 1653-1658) and is a vaccine candidate. The purified cytotoxin
is a protein of 87-94 kD that can be purified in very small amounts
from bacterial culture supernatant.
Material and Methods
Bacterial Strain.
The Helicobacter pylori CCUG 17874 (type strain, Culture
Collection, University of Goteborg) was used.
Media and Supplements.
Brucella Broth (tryptone 10 g l.sup.-1, peptamin 10 g l.sup.-1,
dextrose 1 g l.sup.-1, yeast extract 2 g l.sup.-1, sodium chloride
5 g l.sup.-1 and sodium bisulfite 0.1 g l.sup.-1) (BB)(Difco)
supplemented with 2 g l.sup.-1 of (2,6 di-0-methyl)-b-cyclodextrin
(CD) (Teijin Lim. Tokyo, Japan) and 20 mg/L of streptomycin was
used as liquid medium for comparison purpose. Hysoy or Soytone were
used at a concentration of 10 g/L instead of tryptone and peptamin
present in BB.
Preservation.
Frozen aliquots for inocula were prepared from flask cultures of
2.times.10.sup.8 CFU ml.sup.-1 diluted 1:2 with a solution composed
of glycerol 40%, fetal calf serum (FCS) (HyClone, Logan, Utah) 20%
and 0.4% CD. The suspension obtained was distributed in 3 ml vials
and stored at -80.degree. C. and used as starting frozen vials for
comparison with new frozen vials prepared substituting FCS with
Soytone 20%.
Growth in Liquid Medium.
Initial cultures were performed in 500 ml Erlenmeyer flasks
containing 100 ml of liquid medium. Cultures were inoculated with 3
ml of frozen stocks and incubated at 36.degree. C. for 36 hours
with shaking (100 rpm, 2.5 cm throw) in a microaerobic environment.
Flasks were placed inside an anaerobic jar where BBL Campy Pak
envelopes (Becton Dickinson) were used to generate the proper
conditions. These cultures were then used to inoculate 1000 ml
flasks containing 250 ml of medium and incubated in the same
conditions mentioned above and used to inoculate the
bioreactors.
Culture Vessels and Growth Conditions.
Batch fermentations were carried out in 7 liters bioreactors (MBR
Bioreactors AG, 8620 Wetzikon, C H) containing 5 l of medium. All
cultures were grown at 36.degree. C. The pH values were not
controlled. The dissolved oxygen tension (DOT) was maintained
automatically at the pre-set level (3%) by a two step procedure.
First, air flow rate was increased from 0.1 up to 0.5 l l.sup.-1
min.sup.-1 to satisfy the increasing O.sub.2 demand of the culture.
If further increases were necessary, they were obtained by
supplying pure O.sub.2 up to a maximum of 0.4 l l.sup.-1
min.sup.-1. During the first 12 hours of growth, a constant flow of
N.sub.2 and CO.sub.2 was maintained equal to 0.2 l l.sup.-1
min.sup.-1 and 0.02 l l.sup.-1 min.sup.-1 respectively. The
agitation speed was maintained at 130 rpm. The agitator shaft was
equipped with two Rhuston turbines having a diameter of 7 cm and
the diameter of the bioreactor was 17 cm.
Glucose Feed.
A 50% glucose solution was added at time 0 to give a final
concentration of 5 g/L. Another addition of 5 g/L was made when the
OD was in a range 2-3.
Biomass Determination.
Growth was monitored by optical density at 590 nm against a water
blank (Perkin Elmer 35 spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
Analysis of the VacA Protein.
At determined time points during the fermentation, culture samples
were centrifuged (Biofuge A, Heareus) at 8,300.times.g per 10 min.
The supernatants were precipitated with trichloroacetic acid and
subjected to 9% SDS-Page using a BioRad Mini Protean II apparatus.
Proteins were transferred to nitrocellulose filters (Schleicher
& Schuell) and then incubated overnight with polyclonal
antisera raised against the VacA protein (Telford, J. L. et al.,
(1994), J. Exp. Med., 179, 1653-1658). After incubation for 2 hours
with a horseradish-peroxidase conjugated secondary antibody
(Sigma), the immunoreactive bands were visualized by
4-chloro-naphtol staining.
Results
Brucella Broth is a complex medium composed of tryptone 10 g
l.sup.-1, peptamin 10 g l.sup.-1, dextrose 1 g l l.sup.-1, yeast
extract 2 g l.sup.-1, sodium chloride 5 g l.sup.-1 and sodium
bisulfite 0.1 g l.sup.-1. This medium has been described in many
articles as capable of supporting the growth of H. pylori only when
supplemented with blood derivatives (Cover, T. L. and Blaser, M. J.
(1992), J. Biol. Chem., 267, 10570-10575; Shahamat, M. et al.,
(1991), J. Clin. Microbiol., 29, 2835-2837; Buck, G. E. and Smith,
J. S. (1987), J. Clin. Microbiol., 25, 597-599; and Morgan, D. R.
et al., (1987), J. Clin. Microbiol., 25, 2123-2125). A substantial
simplification of H. pylori growth media was obtained recently when
it was discovered that cyclodextrins could be used in the place of
blood derivatives (Olivieri, R. et al., (1993), J. Clin.
Microbiol., 31, 160-162). The growth of H. pylori using this
simplified medium and glucose feed is reported in FIG. 1. Glucose
feeds were used to ensure that the carbon and energy source was
always present in the medium.
When H. pylori was cultured in these conditions, the production of
VacA in the medium was measured as described above. The results are
shown in FIG. 5 and FIG. 6.
The use of Soytone and Hysoy in the growth media gave the results
reported in FIGS. 2 and 3 respectively. VacA production is reported
in FIG. 5 when Soytone was used and in FIG. 6 when Hysoy was
used.
The results show improvements to the growth media and fermentation
conditions of Helicobacter pylori growth and in the production of
the vacuolating cytotoxin (VacA).
Example II
Haemophilus influenzae are small, non motile, gram negative
bacteria that are the major cause of bacterial meningitidis in
children. These microorganisms are primarily invasive rather than
toxigenic. They are inhabitants of the respiratory tract (commensal
as well pathogenic) and they have antiphagocytic polysaccharides
capsules.
Materials and Methods
Bacterial Strain
Haemophilus influenzae B ATCC 10211
Media and Supplements
The preparation of the media, involves use of different solutions
as follows:
TABLE-US-00001 "Franz A medium" Preparation Component Amount per
liter Purified water 800 mL Glutamic acid 1.6 g +/- 0.01 g
Na2HPO.sub.4 12H2O 5.03 g +/- 0.05 g KCl 0.892 g +/- 0.008 g NaCl
6.005 g +/- 0.06 g NH4Cl 1.25 g +/- 0.01 g Purified water QS to 1.0
liter 3N NaOH as required for pH = 8.2.
The above components are dissolved with mixing. 3N NaOH is used to
pH the solution to pH=8.2
TABLE-US-00002 Ultrafiltered Soytone preparation Component Amount
per liter Soytone 33.3 g +/- 0.03 g Purified water QS to 1.0
liter
This solution was ultrafiltered through a 30 kD TFF apparatus.
TABLE-US-00003 Ultrafiltered Proteose Peptone Component Amount per
liter Proteose peptone 33.3 g +/- 0.03 g Purified water QS to 1.0
liter
This solution was ultrafiltered through a 30 kD TFF apparatus
TABLE-US-00004 Ultrafiltered YE preparation Component Amount per
liter yeast extract 100 g +/- 0.02 g Purified water QS to 1.0
liter
The yeast extract used was HY YEST available from Quest. The
solution was ultrafiltered through a 10 kD TFF apparatus.
TABLE-US-00005 50% Glucose Solution Component Amount per liter
glucose (anhydrous) 500 g +/- 5 g Purified water QS to 1.0
liter
TABLE-US-00006 NAD 0.1% solution Component Amount per liter NAD 1.0
g +/- 0.005 g TRIS 1.21 g +/- 0.01 g Purified water QS to 1.0 liter
HCl 37% as required to pH 7.4 .+-. 0.2
TABLE-US-00007 Hemin 0.4% solution Component Amount per liter Hemin
4 g +/- 0.02 g 0.2N NaOH QS to 1.0 liter
The hemin used is preferably chemically synthesised. Chemically
synthesised hemin is commercially available from Fluka.
550 ml of Franz A was mixed with 450 ml of ultrafiltrated soytone
or Proteose Peptone to obtain one liter of Hib basal medium which
was sterilized by autoclaving at 121.degree. C. for 30 minutes.
After cooling, 10 ml/L glucose solution, 20 ml/L of YE, 2 ml/L NAD
solution, sterilized by filtration, were added (with the additions
mentioned before) and the medium called "Hib Complete medium".
Growth in Liquid Media
Pre-warmed, unbaffeled shake flasks (500/150) were inoculated with
1.0 mL of a working stock vial each. The shake flasks were placed
in an (1 inch throw) incubator-shaker at 35.+-.1.degree. C. at 150
RPM for 6 hours.
After 6 hours, the appropriate amount of the shake flask was
transferred into one 2 L liter unbaffeled shake flask containing
0.5 L of pre-warmed "Hib complete medium". The shake flask was
placed in an (1 inch throw) incubator-shaker at 35.+-.1.degree. C.
at 200 rpm for 9 hours then the content was transferred into a
sterile inoculation and the inoculate then transferred to the
fermenter.
Culture Vessels and Growth Conditions
Batch fermentations were carried out in 30 liters bioreactors (MBR
Bioreactors A G, 8620 Wetzikon, C H) containing 20 l of medium.
The cultures were growing at 35.degree. C. and 2 psi back pressure.
The pH was controlled to 7.3 with 3 N NaOH. The initial agitation
rate was set at minimum of 150 rpm, and bottom aeration at 10
L/minute. DOT was maintained at 35% by rpm control in a range
150-400, then supplementing with oxygen if necessary. Antifoam was
added manually to control foaming. Residual concentration of
glucose was detected and when it was around 2 g/L, 0.2 liters of
glucose solution were added.
Biomass Determination.
Growth was monitored by optical density at 590 nm against a water
blank (Perkin Elmer 35 spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
Analysis of Hib PS.
This analysis was performed by Rocket immuno electrophoresis as
described by Weeke, B., Scand. J. Immunol. 2, 37-46, (1973), herein
incorporated by reference.
Results
The growth curves obtained with Soytone and with Proteose peptone
are compared in FIG. 7. The yield of Hib PS was 600 mg/L and 150
mg/L in the media containing Soytone and Proteose peptone
respectively. Although the growth curves are quite similar using
the two media, there is a fourfold increase in the yield of Hib PS
using Soytone. The use of the culture media containing the
vegetable derived proteinaceous material leads to an increased
yield of polysaccharides, such as Hib PS, compared to the use of a
culture medium containing animal derived proteinaceous
material.
Example III
C. diphtheriae are gram positive, rod like microorganisms, which
arrange themselves in palisades. C. diphtheriae lysogenization by a
bacteriophage causes the synthesis of a potent toxin whose
expression is regulated by iron concentration.
Materials and Methods
Bacterial Strain
C. diphtheriae CN 2000
Media and Supplements
The preparation of the media, involves use of different solutions
as follows:
TABLE-US-00008 A. Yeast Extract (YE) and Casamino Acids (CAA)
Ultrafiltered Component Amount g/L Purified water 800 ml Yeast
Extract 20 g/L Casamino Acids 10 g/L Purified water QS to 1 L
This solution was ultrafiltered through a 10 kD TFF apparatus and
the permeate added to the deferration vessel.
TABLE-US-00009 A.A Yeast Extract (YE) and Soytone Ultrafiltered
Component Amount g/L Purified water 800 ml Yeast Extract 20 g/L
Soytone 10 g/L Purified water QS to 1 L
This solution was ultrafiltered through a 10 kD TFF apparatus and
the permeate added to the deferration vessel.
TABLE-US-00010 A.B Yeast Extract Ultrafiltered Component Amount g/L
Purified water 800 ml Yeast Extract 30 g/l Purified water QS to 1
L
This solution was ultrafiltered through a 10 kD TFF apparatus and
the permeate added to the deferration vessel.
A.1 Deferration
The following components were introduced into an agitated
vessel
TABLE-US-00011 Component Amount UF (YE + CAA) solution 1 L
KH.sub.2PO.sub.4 5 g/L CaCl.sub.2--2H.sub.2O, 50% (w/v) 2 mL/L
L-tryptophan, 1% (w/v) 5 mL/L (0.05 g/L) 3N NaOH (enough to correct
pH to 7.4)
UF indicates that the solution is ultrafiltered.
With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with CAA".
A.1.A Deferration
The following components were introduced into an agitated
vessel
TABLE-US-00012 Component Amount UF (YE + Soytone) solution 1 L
KH.sub.2PO.sub.4 5 g/L CaCl.sub.2--2H.sub.2O, 50% (w/v) 2 mL/L
L-tryptophan, 1% (w/v) 5 mL/L (0.05 g/L) 3N NaOH (enough to correct
pH to 7.4)
With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with Soytone".
A.1.B Deferration
The following components were introduced into an agitated
vessel
TABLE-US-00013 Component Amount UF YE solution 1 L KH.sub.2PO.sub.4
5 g/L CaCl.sub.2--2H.sub.2O, 50% (w/v) 2 mL/L L-tryptophan, 1%
(w/v) 5 mL/L (0.05 g/L) 3N NaOH (enough to correct pH to 7.4)
With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with YE only".
B. "Supplements" Solution
TABLE-US-00014 B1) Solution A Component Amount per liter
MgSO.sub.4--7H.sub.2O 225 g Beta-alanine 1.15 g Nicotinic acid 1.15
g Pimelic acid 0.075 g CuSO.sub.4 0.50 g ZnSO.sub.4--7H.sub.2O 0.40
g MnCl.sub.2--4H.sub.2O 0.15 g HCl, 37% 30 mL Purified water QS to
1.0 liters
TABLE-US-00015 B2) Solution B Component Amount per liter Purified
water 800 mL L-cystine 200 g HCl, 37% 200 mL
The solutions are mixed individually for 10 minutes. After
dissolution, 20 mL of Solution A and 10 mL of Solution B are mixed
and filtered through a 0.2 micron filter. The solution is stored at
4.degree. C. covered from light.
Flasks Sterilization
Once deferrated, filtered media (A.1 or A.1.A or A.1.B) are loaded
into the flasks. Sterilize the flasks for 25 minutes at 121.degree.
C.
TABLE-US-00016 Post-Sterilization Adjustments 1. Add "50% Maltose"
30 ml/L 2. Add "Supplements" 3.0 ml/L
Media with 1+2 above: "Complete CY medium with CAA or with Soytone
or with YE only" Growth in Liquid Media
500 ml unbaffeled shake flasks each with 100 ml of medium, were
inoculated with 1.0 mL of a working stock vial.
The shake flasks were placed in an (1 inch throw) incubator-shaker
at 35.+-.1.degree. C. at 100 rpm for 5 hours. After 5 hours, the
agitation is increased to 250 rpm for 43 hours.
The process has two distinct phases: 1) Exponential growth), and 2)
Production phase. The transition from the two phases is gradual and
is marked by reductions in cell growth rate and oxygen demand.
Biomass Determination.
Growth was monitored by optical density at 590 nm against a water
blank (Perkin Elmer 35 spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
Results
After 48 hours of incubation, the OD and the Lf/ml in the flasks
were:
TABLE-US-00017 medium with CAA. OD = 7.5 Lf/ml = 50 medium with
Soytone. OD = 7.87 Lf/ml = 60 medium with YE only OD = 8.07 Lf/ml =
60
Example IV
Neisseria meningitidis are non motile gram negative cocci, most
often growing in pairs but occasionally in tetrads or clusters.
They have antiphagocytic polysaccharides capsules which is the
basis for serogroups.
Materials and Methods
Bacteria Strain
Neisseria meningitidis. C11
Media and Supplements
Preparation of the media involves use of different solutions as
follows:
TABLE-US-00018 "Franz medium" Preparation Inoculum Shake Flask
Medium Preparation Component Amount per liter Purified water 800 mL
Glutamic acid 1.6 g Na2HPO.sub.42H2O 15.5 g KCl 0.09 g NH4Cl 1.25 g
Purified water QS to 1.0 liter 3N NaOH as required for pH = 7.6
Dissolve the above components with mixing. Sterilize by autoclaving
at 121.degree. C., 30 minutes. After cooling, the "50% Glucose" and
"Men C Supplements" are added as below.
TABLE-US-00019 "50% Glucose" 10 mL/L +/- 0.3 mL "Men C Fermentation
Supplements" 20 mL/L +/- 0.3 mL
With the addition of the glucose and supplements solutions, the
medium can be called "Franz Complete medium".
Preparation of the 20 L Fermenter
A. The Following Components Must be Introduced to a Mixing Vessel
and Dissolved to Create 20 L of Watson Base Medium with CAA:
TABLE-US-00020 Component Amount per liter Purified water 800 mL
Glutamic acid 1 g +/- 0.01 g Na.sub.2HPO.sub.42H.sub.2O 3.25 g +/-
0.03 g KCl 0.09 g +/- 0.001 g Casamino acid 10 g +/- 0.1 g Purified
water QS to 1.0 liter 3N NaOH as required for pH = 7.6
A.A The Following Components Must be Introduced to a Mixing Vessel
and Dissolved to Create 20 L of Watson Base Medium with Amisoy:
TABLE-US-00021 Component Amount per liter Purified water 800 mL
Glutamic acid 1 g +/- 0.01 g Na.sub.2HPO.sub.42H.sub.2O 3.25 g +/-
0.03 g KCl 0.09 g +/- 0.001 g Amisoy 10 g +/- 0.1 g Purified water
QS to 1.0 liter 3N NaOH as required for pH = 7.6
B. Fermenter Sterilization
Sterilize the fermenter for 25 minutes at 121.degree. C. After
sterilization, set the following conditions: temperature,
35.degree. C.; aeration at 10 L/minute; agitation at 150 rpm, and
backpressure at 0.2 bar. After cooling the fermenter to 35.degree.
C., and take a sample to measure pH, adjust the fermenter pH probe
calibration and then adjust the medium pH to 7.6.
TABLE-US-00022 C. Post-Sterilization Adjustments 1. Add "50%
Glucose" 10 ml/L 2. Add "Men C Supplement" 20 ml/L Medium with 1 +
2 above: "Watson Complete medium"
TABLE-US-00023 A. "50% Glucose" Solution Component Amount per liter
glucose (anhydrous) 500 g +/- 5 g Purified water QS to 1.0 L
This solution is sterilized by autoclaving at 121.degree. C. for 30
minutes.
B. "Men C Supplements" Solution
TABLE-US-00024 B.1 Ultrafiltered YE Component Amount Purified water
QS to 1 L Yeast Extract 125 g/L
This solution is ultrafiltered through a 10 kD TFF apparatus.
The retentate is discarded after the requisite permeate has been
collected, and the permeate is added to the vessel containing the
supplement.
TABLE-US-00025 B.2 Component Amount per liter of UF YE
MgSO.sub.4--7 H.sub.2O 30 g +/- 0.5 g L-cystein-HCl 1.5 g +/- 0.2
g
Add the chemicals to 1 L UF YE, mix for 10 minutes, filter
sterilized through a 0.2 micron filter and transfer to the
fermenter.
C. 3 NNaOH 3N NaOH=12%(w/v)NaOH=120 g/L NaOH D. Antifoam
The antifoam used is Dow Corning 1510. It is sterilized by
autoclaving at 121.degree. C. for 30 minutes
Growth in Liquid Media
A 500 milliliter flask is inoculated with 1.0 mL of a working stock
vial. The shake flask contains 150 mL of complete "Franz
medium".
The inoculated shake flasks is placed in an (1 inch throw)
incubator-shaker at 35.+-.1.degree. C. at 150 rpm. After 10 hours,
the flask is aseptically sampled. The optical density should be
between 1.3-3.3 (at 590 nm) if so, the four 2 L shake flasks are
each inoculated with the appropriate volume from the 150 mL shake
flask.
Each 2 L shake flask contains 0.2 L of pre-warmed "Franz Complete
medium".
The shake flasks are placed in an (1 inch throw) incubator-shaker
at 35.+-.1.degree. C. at 200 rpm. After 10 hours, the contents of
each shake flask are transferred into the sterile inoculation can
and inoculate the fermenter.
Culture Vessel and Growth Conditions
Batch fermentations were carried out in 30 liters bioreactors (MBR
Bioreactors A G, 8620 Wetzikon, C H) containing 20 L of medium.
The cultures were growing at 35.degree. C. and 14.times.10.sup.3
N/m.sup.2 (2 psi) back pressure. The pH was controlled to 7.3 with
3 N NaOH. The initial agitation rate was set at minimum of 150 rpm,
and bottom aeration at 10 L/minute. DOT was maintained at 35% by
rpm control in a range 150-400, then supplementing with oxygen if
necessary. Antifoam was added manually to control foaming. Residual
concentration of glucose was detected and when it was around 2 g/L,
0.2 liters of glucose solution were added.
Biomass Determination.
Growth was monitored by optical density at 590 nm against a water
blank (Perkin Elmer 35 spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
Analysis of MenC PS
Quantitative estimation of the polysaccharides was performed
analyzing the sialic acid content according to the method reported
in Biochimica and Biophysica Acta (1957), 21, 610, by Lars
Svennerholm.
Results
The growth curves obtained with Amisoy and with Casamino acids are
compared in FIG. 8. The yield in MenC PS was 307 mg/L and 345 mg/L
in the media containing Amisoy and Casamino acids respectively. The
growth curves and PS production are quite similar using the two
media.
It will be understood that the invention is described above by way
of example only and modifications may be made within the scope of
the invention as defined in the appended claims.
Example V
Clostridium tetani is a slender bacillus measuring 2 .mu.m in
length and 0.3-0.5 .mu.m in width. It often exists in the form of a
rather long filament-like cell. When spores are formed, the
bacillus assumes the characteristic drumstick appearance. It is a
mobile organism, gram positive, but its gram stain can become
variable or even negative in aging cultures. Clostridium tetani is
a strict anaerobe and produces two exotoxins. One of these, the
tetano spasmin, is a neurotoxin responsible for the whole clinical
picture of the disease.
Materials and Methods
Bacterial Strain
Clostridium tetani Harvary Y-VI-3.
Media
The seed cultures were prepared using the medium reported below
expressed in g/L:
TABLE-US-00026 Component Amount g/L N-Z Soy 15.0 Glucose 5.5 Yeast
extract 5.0 NaCl 2.5 L-Cysteine 0.5 Sodium thioglycollate 0.5 Agar
0.75 PH = 7.1
The production media were prepared as modification of the
Mueller-Miller medium described in the WHONSQ/GEN/94 (1990). In
this medium, beef heart infusion and casein solution are used, in
the modified media reported below, expressed in g/L, Hysoy and
Soytone were used instead of beef heart infusion and casein
solution:
TABLE-US-00027 Component Amount g/L Glucose-H.sub.2O 12.1 NaC1 2.5
Na.sub.2HPO.sub.4--12H.sub.2O 2.5 KH.sub.2PO.sub.4 0.15
MgSO.sub.4--7H.sub.2O 0.15 Amino acids solution 17.5 ml Vitamine
solution 4.2 ml NaOH 5M 4.0 ml FeSO.sub.4--7H.sub.2O (1% sol.) 4.0
ml Soy derivatives 20.0 PH = 7.3
Sterilised by autoclaving at 120.degree. C. for 20 min
Amino Acids Solution:
TABLE-US-00028 Component L-Tyrosine 28.51 g/L Uracil- 0.142 g/L
L-Cystein- 14.25 g/L HCl 37% 131.6 ml/L
Vitamin Solution:
TABLE-US-00029 Component Ca pantithenate 238.1 mg/L Thiamine 59.7
mg/L Pyridoxin 59.7 mg/L Riboflavin 59.7 mg/L Biotin 0.73 mg/L
Ethanol 256.4 ml/L
Growth in Liquid Media
Two 25 ml tubes, containing 15 ml of seed medium, were inoculated
with 0.5 ml each of working seed vial and incubated at 35.degree.
C. for 29 hrs in anaerobic jar where a Gas generating kit (OXOID)
was used. A second series of tubes were inoculated by 1.5 ml of the
first tubes and incubated in the same conditions reported above for
24 hrs.
7 ml of these cultures were used to inoculate 100 ml tubes
containing 75 ml of the same medium. These tubes were incubated in
the same conditions reported above for 24 hrs.
The entire content of these tubes were used to inoculate 5000 ml
beakers containing 2500 ml of the production medium.
Biomass Determination
Growth was monitored by optical density at 590 nm against a water
blank (Pharmacia spectrophotometer), light path of 1 cm. Purity
checks of the samples were made by Gram staining.
Results
After 186 hrs of incubation, the OD and the Lf/ml in the breakers
were:
TABLE-US-00030 Medium with Hysoy OD = 1.08 Lf/ml = 60 Medium with
Soytone OD = 0.74 Lf/ml = 60 Medium with beef heart infusion and
casein OD = 1.236 Lf/ml = 60 solution
Example VI
Bordetella pertussis is a gram negative coccobacillus about 0.5
.mu.m in diameter and 0.5 to 2 .mu.m in length. Its nutritional
requirements are simple, and it does not utilize sugars. It is
extremely sensitive to fatty acids and survives poorly without
protective factors.
Materials and Methods
Bacterial Strain
Bordetella pertussis 9K/129G (Pizza, M., et al. (1989) Science,
246, 497-500).
Media
The seed and production cultures were prepared using CL medium
(Imaizumi, A., et al. (1983) Infection and Immunity, 41 (3),
1138-1143) reported below expressed in g/L:
TABLE-US-00031 Component Amount g/L Sodium L-glutamate 10.7
L-proline 0.24 NaC1 2.5 KH.sub.2PO.sub.4 0.5 KCl 0.2
MgCl.sub.2--6H.sub.2O 0.1 CaCl.sub.2 0.02 Tris 6.1 L-Cysteine* 0.04
FeSO.sub.4--7H.sub.2O* 0.01 Niacin* 0.004 Glutathione reduced* 0.15
Ascorbic acid* 0.4 Casaminoacid 10.0 Dimethyl-B-cyclodextrin 1.0 PH
adjusted to 7.6 with HCl. *sterilized by filtration and then added
aseptically to the autoclaved medium The modified medium contained
10 g/l of N-Z soy instead of Casaminoacid.
Growth in Liquid Media
500 ml unbaffeled shake flasks each with 100 ml of CL medium or the
modified one, were inoculated with 3.0 mL of a working stock
vial.
The shake flasks were placed in an (1'' throw) incubator-shaker at
35.+-.1.degree. C. at 100 RPM for 12 hours. After 12 hours, the
agitation was increased to 250 RPM for another 16 hours.
Biomass Determination
Growth was monitored by optical density at 590 mm against a water
blank (Pharmacia spectrophotometer), light path of 1 cm. Purity
checks of the samples were made by Gram staining.
Analysis of PT
This analysis was performed by ELISA (Nencioni, L., et al. (1990)
Infect. Immun., 58, 1306-1315).
Results
After 28 hrs of incubation, the OD and PT (mg/L) in the flasks
were:
TABLE-US-00032 medium with CAA. OD = 2.31 PT = 2.4 mg/L Medium with
NZ-soy. OD = 1.99 PT = 2.25 mg/L
* * * * *